The invention relates to pharmaceutical compositions with activity against peptic ulcer containing a zinc associate (complex) of hyaluronic acid as well as a process for the preparation thereof.
The invention furthermore relates to the use of the zinc associate (complex) of hyaluronic acid, i.e. zinc hyaluronate for the preparation of pharmaceutical compositions of activity against peptic ulcer and a method for the treatment and prevention of peptic ulcer. The invention also relates to the use of zinc hyaluronate for the prevention of reinfection after the healing of peptic ulcer.
The macromolecule known as hyaluronic acid usually occurring in the form of its sodium salt, is a compound known for more than 50 years. It had first been described by Meyer et al [J. Biol. Chem. 107, 629 (1934)]. Hyaluronic acid is a highly viscous native glucosaminoglycan containing alternating xcex21-3 glucuronic acid and xcex21-4 glucosamine moieties, its molecular weight is between 50000 and several millions. Hyaluronic acid is found in the connective tissues of all mammals; it occurs in higher levels in the skin, vitreous body of the eye, synovial fluid, umbilical cord as well as cartilaginous tissue.
Up to recently, hyaluronic acid has been employed as the sodium salt in therapyxe2x80x94mainly in ophthalmology, dermatology, surgery, articular therapyxe2x80x94and cosmetics. The salts of hyaluronic acid formed with alkali, alkaline earth, magnesium, aluminum, ammonium and substituted ammonium ions may serve as carriers for promoting the absorption of drugs (see the Belgian patent specification No. 904,547). Heavy metal salts of hyaluronic acid, among these the silver salt, are employed as fungicides; whereas the gold salt is useful for the treatment of rheumatoid arthritis (see the patent specification No. WO 87/05517). However, severe adverse effects of silver and gold compounds are known, i.e. their effects on the immune system, hematopoietic organs and nervous system [M. Shinogi, S. Maeizumi: xe2x80x9cEffect of preinduction of metallothionein on tissue distribution of silver and hepatic lipid peroxidationxe2x80x9d, Biol. Pharm. Bull. (Japan), April 1993 16 (4), p. 372-374; C. Masson et al.: Rev. Med. Interne (France) May-June 1992, 13 (3) p. 225-232 (1992)].
Associates (complexes) of deprotonated hyaluronic acid with 3d metal ions of 4th period of the Periodic Table such as zinc and cobalt hyaluronates with a curative effect especially on crural ulcer, decubitus ulcer or the like are discussed in the Hungarian patent specification No. 203,372, with confirmations by external use.
Now, it has been found that the zinc associate (complex) of hyaluronic acid, i.e. zinc hyaluronate possesses significant gastroprotective activity so it can be used in the prophylaxis and treatment of peptic ulcers including Helicobacter pylori-induced ulcers.
There are known structurally similar gastroprotective compounds in the literature. The anti-gastric ulcer effect of zinc salts of acidic polysaccharides and within these, of mucopolysaccharides is described in the published Japanese patent application No. 60-48950, giving a list of alga-derived agar agaropectin of marine origin, carrageenin, alginic acid as acid polysaccharides; hyaluronic acid, heparin, chondroitin sulfate as mucopolysaccharides; as well as other compounds such as dextran sulfate, carboxymethylcellulose and the like and pectinic acid of plant origin. However, according to this application, the molecular weights of acidic polysaccharides are about a few thousands, preferably about 20 thousand. The zinc salt of hyaluronic acid and its pharmacological activity are not published in the examples at all.
The U.S. Pat. No. 5,514,660 describes the antiulcer effect of pharmaceutical compositions containing an oligosaccharide type active ingredient, together with examples of investigations on the effects of the compounds on the Helicobacter pylori-induced ulcer. Neither hyaluronic acid nor its zinc associate are involved in this specification and no wound-healing effect is mentioned.
Peptic ulcer disease is a complex and multifactorial disease which concerns a great part of civilized population. Many questions still remain in relation to the exact pathogenesis of this disease. In the last five years a new approach has emerged due to the xe2x80x9cre-discoveryxe2x80x9d of Helicobacter pylori. It is generally accepted that the development of peptic ulcer disease in humans is associated with the infection of Helicobacter pylori, but on the other hand H. pylori can not be held responsible for all causes of peptic ulcer. Up to now the peptic ulcer therapy has not changed essentially: still H2 blockers and proton pump inhibitors are the most important requisites of the therapeutic arsenal, whilst the possible therapeutical methods for killing the H. Pylori bacteria are now disclosed in the literature. In addition, the treatment of gastro-duodenal damage caused by nonsteroidal anti-inflammatory drugs has become the center of interest.
A peptic ulcer, developed by any mechanism, may be characterized by an upset of that balance, which exists under healthy conditions between aggressive factorsxe2x80x94which induce development of ulcersxe2x80x94and defensive factorsxe2x80x94which protect the stomach against induction of ulcers. Thus, an ulcer develops in each case when the aggressive factors are enhanced beyond certain limits and/or the defensive factors become weaker. Defensive factors include the resistance of gastric mucosa, sufficient blood supply of gastric mucosa, and mucus formation [H. Shay: Etiology of peptic ulcer, Am. J. Dig. Dis. 6, 29-49 (1961)]. Among the aggressive factors, the leading ones are the secretion of hydrochloric acid and pepsin as well as all factors stimulating the secretion of any of both substances. The secretion of acid may be enhanced also by a pathological vagal stimulus or increased gastrin formation, by an autoimmune mechanism and in some cases by hormonal effect. In addition, the balance between aggressive and defensive factors may be disturbed also by injuries affecting the whole body. Thus, peptic ulcer is a multifactorial disease. Agents used in ulcer therapy are intended to reduce the role of aggressive factors and/or to enhance the role of defensive factors. Up to recently, the pharmacological investigations have sought ways to weaken the effects of aggressive factors so the primary target was to moderate the acid-pepsin activity. Drugs used in the treatment of ulcer were targeted first to neutralize the acid (antacids such as sodium hydrogen carbonate, or aluminum hydroxide) or to inhibit its secretion (H2 blockers, e.g. cimetidine, famotidine, proton pump inhibitors, e.g. omeprazole); only recently research into those agents which enhance the role of defensive factors became important, beyond the development of a monotherapy useful for killing Helicobacter pylori.
However, compositions strengthening the defensive factors are available only in a limited number and they show some side effects. Such compositions are colloidal bismuth subcitrate (CBS, DeNol), sucralfate and misoprostol. CBS contains bismuth, which is responsible for toxicological problems. Sucralfate a basic aluminum salt of sulphated sucrose possesses some adverse effects, e.g. nausea, vomiting, aluminum intoxication, etc., and the misoprostol, a synthetic prostaglandin analogue, induces an enhanced intestinal activity or nausea.
It can be seen from the above that there is a further need for effective and safe drugs to treat ulcers not accompanied with an increased acid secretion. Such compositions could be employed with success in cases where the aim is to prevent the gastric lesions from the gastric irritative, damaging effect of e.g. nonsteroidal anti-inflammatory drugs such as indomethacin, aspirin by strengthening the mucosal protective mechanism. The number of patients having peptic ulcers due to NSAID (nonsteroidal anti inflammatory drug) consumption might grow in the future if we take into consideration the data showing that more than ten million tablets of nonsteroidal anti-inflammatory drugs are consumed daily worldwide forming the group of most widely used medicaments of today.
The reinforcement of protective mechanism can be important for ulcers caused by Helicobacter pylori infection, too, since the bacteria produce a number of toxins and enzymes (urease, protease, catalase, lipase) damaging the gastric mucosa and giving a free way for the acid and pepsin towards gastric epithelium.
In order to meet the demand described above our aim was to focus on the treatment of peptic ulcer by examining the assumed gastroprotective activity of zinc hyaluronate, a zinc associate (complex) of hyaluronic acid. According to our experimental results, zinc hyaluronate shows a considerable gastroprotective action and seems to be very useful in field of action mentioned above, i.e. in the prevention (prophylaxis) of peptic ulcer formation and/or in the treatment of a developed ulcer, respectively.
An object of the invention is to provide pharmaceutical compositions with anti-peptic ulcer effect, containing zinc associate (complex) of hyaluronic acid i.e. zinc hyaluronate as active ingredient; as well as to a process for the production thereof by mixing the zinc hyaluronate active ingredient (prepared in a known manner) with therapeutically useful carriers and/or additives and converting the mixture to a pharmaceutical composition.
Another object of the invention is the use of the zinc associate (complex) of hyaluronic acid for the preparation of pharmaceutical compositions with activity against peptic ulcer and a method for the treatment and prevention of peptic ulcer.
The gastroprotective effect of zinc hyaluronate was verified by various pharmacological investigations. Zinc hyaluronate solutions according to Hungarian patent specification No. 203,372 were used for these examinations. Sodium hyaluronate and zinc chloride, compounds from which zinc hyaluronate was prepared, were also investigated. The molecular weight at the zinc hyaluronate ranges from 500.000 to 1.200.000 daltons.
In pharmacological experiments used to prove the gastroprotective effect of zinc hyaluronate, he investigated how the effectiveness changed with the modification of its molecular weight, and with the various grades of purities. For this purpose, solutions of zinc hyaluronate(I), zinc hyaluronate(II), and zinc hyaluronate (III) with molecular weights of 750.000, 900.000 and 1.200.000, respectively were tested, having the degree of highest purity (xe2x80x9cpurissimumxe2x80x9d) in the experiments. Zinc hyaluronate(M) was of xe2x80x9cpurumxe2x80x9d quality degree that is prepared from a low-graded quality material. Our last aim was to make comparison between zinc hyaluronate and one of the most widely used gastroprotectants, sucralfate.
It is evident for a person skilled in the art that, having mixtures of molecules with various degrees of polymerization and molecular weights between very wide limits such as hyaluronic acid compounds, they cannot be characterized by an exact value of molecular weight. In the case of zinc hyaluronate, similarly as with all other hyaluronic acid compounds, xe2x80x9cmolecular weightxe2x80x9d is meant a mean value of molecular weights shown by a mixture of molecules with various degrees of polymerization measured by a given method of molecular weight determination.
The zinc hyaluronate associates of various molecular weights were dissolved in an aqueous solution of 1.0% for the pharmacological experiments. The following example described the preparation""s procedure of 1.0% solution of zinc hyaluronate. If not noted otherwise, the percentages mean weight/volume% values.
Sodium hyaluronate (1.0 g) is swollen in 30 ml of water while constant stirring then after adding 18.75 ml of 0.1 molar zinc chloride solution it is filled to 100 ml with distilled water.
The characteristics of sodium hyaluronate used for the preparation of solutions of zinc hyaluronate with various molecular weights and degrees of purity employed in our pharmacological investigations are summarized in the following Table.
For the examinations, the employed doses of solutions containing zinc hyaluronate with various molecular weights and degrees of purity, as well as of sodium hyaluronate and zinc chloride solutions were diluted with distilled water and administered in a volume of 10 ml/kg of body weight. The doses of sucralfate used as reference substance as well as indomethacin and powdered charcoal were suspended in 1-2 drops of Tween 80 and diluted to 5 ml/kg of body weight with physiological saline (0.9% by weight/volume of sodium chloride). The doses used of N-ethylmaleimide and NG-nitro-L-arginine methyl ester (L-NAME) were diluted to a volume of 5 ml/kg of body weight. The platelet activating factor (PAF) was dissolved in 0.25% by weight/volume bovine serum albumin prepared with 0.9% by weight/volume sodium chloride solution. The sources of substances employed were as follows.
Zinc hyaluronate (I), (II), (III) and (M) as well as sodium hyaluronate (Gedeon Richter); zinc chloride (Merck); sucralfate (UlcerlminR Chugai); indomethacin (Sigma); N-ethylmaleimide (Fluka); NG-nitro-L-arginine methyl ester (Sigma); L-arginine (free base) (Sigma); D-arginine (free base) (Sigma); bovine serum albumin (Sigma); charcoal (Sigma).
The pharmacological activity of the zinc hyaluronate associate was studied by using the following methods.
1. Inhibition of Acidified-ethanol Induced Gastric Lesions
Female RG-Wistar rats weighing between 120-150 g were used. Before experiments animals were fasted for 24 hours but received water ad libitum.
Experiments were carried out according to the technique similar to that described by A. Robert [Gastroenterology 77, 7661 (1979)].
Acidified ethanol (mixture of 50 ml of abs. ethanol and 1 ml of concentrated HCI) was used as strong irritants. Dose of acidified ethanol was 0.5 ml/100 g body weight with intragastric administration. Test compounds were given orally 30 minutes before the acidified ethanol challenge. When the duration of action of test compound was studied test material was administered 60 or 120 minutes earlier to the irritating effect induced by acidified-ethanol effect. One hour after the acidified ethanol treatment rats were sacrificed by cervical dislocation. Stomachs were removed and opened along the greater curvature and mildly washed down. The longitudinal hemorrhagic lesions were assayed by length.
ED50 value was defined as the dose that gave a 50% decrease in ulcer index.
To study the mechanism of action of the compound to be tested, the involvement of endogenous NO in the development of acidified-ethanol induced gastric damage was also studied by intraperitoneal administration of L-NG-nitroarginine methyl ester (L-NAME). L-NAME at a dose of 25 mg/kg was injected 15 minutes before the test compound. In subsequent experiments the test compound (p.o.), L-NAME (25 mg/kg i.p.), L-arginine (400 mg/kg i.v.) were administered 30, 45 or 60 minutes before oral administration of acidified-ethanol.
The results are summarized in the following Tables. The control group shown in the Tables received only vehicle (distilled water) instead of zinc hyaluronate.
It can be observed from the results shown in Tables 1, 2, 3 and 4 that zinc hyaluronate of various molecular weights and purities possess a significant gastroprotective effect: after oral administration. The compound inhibits in a dose-dependent manner the formation of acidified ethanol induced gastric damage. The ED50 values of various zinc hyaluronates were: zinc hyaluronate(I): 15.9 mg/kg; zinc hyaluronate(II): 11.1 mg/kg; zinc hyaluronate(III): 33.2 mg/kg; and zinc hyaluronate(M): 10.4 mg/kg. In opposition to the zinc hyaluronate associates (I), (II), (III) and (M), sodium hyaluronate given in a 100 mg/kg oral dose exerted a protective action of only 36% on acidified ethanol induced gastric lesion (Table 5). The ED50 value related to zinc chloride is 4.5 mg/kg after oral administration (Table 6). Sucralfate, a known cytoprotective agent used as reference compound showed a weaker activity (with an ED50 value of 112 mg/kg) than the tested zinc hyaluronates did (Table 7).
The duration of the effect was investigated on the zinc hyaluronate associate(M). Increasing the pretreatment time the ED50 value rose (Tables 8 and 9). It can be seen from the results that the zinc hyaluronate(M) does not bind to the gastric mucosa in an irreversible manner.
The connection between the cytoprotective effect of zinc hyaluronate and endogenic NO was studied by using an inhibitor of the nitrogen(II) oxide biosynthesis namely, NG-nitro-L-arginine methyl ester (L-NAME). Considering that zinc hyaluronate(II) and zinc hyaluronate(M) had shown the same activity in acidified ethanol test, the latter was the only one tested in this experiment.
It is obvious from our results (Table 10) that a pretreatment by L-NAME considerably reduced the gastroprotective activity of zinc hyaluronate(M); this was suspended by the pretreatment with L-arginine but was not abolished by pretreatment with D-arginine. On the other hand, the protective effect of zinc chloride was not influenced by a pretreatment with L-NAME in this test (Table 11). Sodium hyaluronate was not tested against L-NAME because the gastroprotective effect of this substance was so mild (Table 5) that the results can not be evaluated exactly. The phenomenon, that the protective effect of zinc hyaluronate is significantly decreased by L-NAME, whereas that of zinc chloride is not affected, seems to prove that NO might play a role in the development of cytoprotective effect of zinc hyaluronate(M).
2. Inhibition of Indomethacin Induced Gastric Lesion Female RG Wistar rats weighing between 120-150 g were used. Before the experiments animals were fasted for 24 hours but received water ad libitum. Test compounds were given orally, 30 minutes later the animals were treated subcutaneously with indomethacin at a dose of 40 mg/kg. Four hours after the irritating indomethacin administration the animals were killed by cervical dislocation. The stomachs were removed, opened along the greater curvature and the hemorrhagic lesions were counted on the glandular stomach.
ED50 value represents the dose which protects the stomach by 50 % from the damaging effect of indomethacin.
The results are shown in the following Tables. The control group shown in the Tables received only vehicle (distilled water) instead of zinc hyaluronate.
It can be seen from the results that zinc hyaluronates (I), (II), (III) and (M) are effective on indomethacin-induced gastric lesion (Tables 12, 13, 14 and 15). It is also evident from the results that zinc hyaluronates inhibit development of indomethacin induced gastric lesions in a dose-depending manner. After oral administration their ED50 values are: 2.8 mg/kg, 5.8 mg/kg, 10.2 mg/kg and 6.2 mg/kg, respectively (see Tables 12, 13, 14 and 15).
It is obvious from the data of Table 16 that sodium hyaluronate and zinc chloride do not provide any effect to protect the gastric mucosa against the damaging action of indomethacin. Furthermore, it can be seen from the higher ED50 value (74.9 mg/kg) of sucralfate (Table 17) that the protective action of zinc hyaluronate exceeds the effect of sucralfate.
3. Inhibition of the Stress Induced Gastric Lesions
Female RG Wistar rats weighing between 120-150 g were used. Before the experiments animals were fasted for 24 hours but received water ad libitum. Experiments were carried out according to the technique similar to that described by Senay et al., (1967). [Proc. Soc. Exp. Biol. Med.124,1221, (1967)].
Based on the results of acidified ethanol tests, where zinc hyaluronate (II) and zinc hyaluronate (M) proved to have identical activity, zinc hyaluronate (M) was only studied in the stress model.
Test compounds were given orally. 30 minutes later the animals were immobilized in plastic restraint boxes then placed into a refrigerator at 4xc2x0 C.-8xc2x0 C. temperature. Four hours later the animals were removed from both the refrigerator and the restraint boxes and one hour later they were sacrificed by cervical dislocation. The stomachs were removed, opened along the greater curvature and hemorrhagic lesions were scored by a zero to 3 scale: stomachs free from any damage were given a score of 0, stomachs with only a few hemorrhagic lesion were given a score of 1, stomachs with more lesion numbers than 10 were given a score of 2, and stomachs with a lot of lesions were characterized with a score of 3.
The ED50 were defined as the dose needed to decrease the score of gastric lesions by 50%. The results can be observed in the following Tables.
Various stress situations, such as cold and considerable restriction of movement induce acute gastric damage in rats. It can be seen from the results, that the cold-restraint stress was prevented by zinc hyaluronate (M) in a dosedependent manner with ED50=29.4 mg/kg in the case of oral administration. (Table 18). On the other hand sodium-hyaluronate and zinc chloride had only slight protective effects on cold-restraint induced gastric damage. (Table 19).
4. Inhibition of the Gastric Damage Induced by the Platelet Activating Factor (PAF)
Male, RG-Wistar rats weighing between 200-250 g were used. Before the experiments animals were fasted for 24 hours but received water ad libitum. Experiments were carried out according to the technique similar to that described by Wallace and Whittle (1986). [Br.J.Pharmac., 89,415, (1986)]. Animals were anaesthetized by intraperitoneal administration of urethane at a dose of 1 g/kg in a volume of 10 ml/kg. Femoral vein was then prepared and cannulated for administration of ulcerogenic PAF. After the surgery PAF was infused intravenously at a rate of 0.1 ml/min for ten minutes at a dose of 200 ng/kg/min. Test compounds were given 30 minutes before the PAF challenge. One hour after the infusion of PAF animals were sacrificed by cervical dislocation; their stomachs were removed and opened along the greater curvature. The hemorrhagic erosions were evaluated by a zero to three scoring system. Stomachs free from any damage were characterized by a score of zero, stomachs with only a few, little change from normal were given a score of 1, stomachs with a moderate damage were characterized by a score of 2, and stomachs with severe, diffuse hyperaemia and/or haemorrhagia were given a score of 3.
PAF (platelet activating factor) is an endogenous phospholipid which has nowadays been described as the most potent gastric ulcerogen. Its endogenic release may play a role in the development of certain forms of peptic ulcer [Rosam et al. (1986); Wallace et al. (1986)].
The results are illustrated in the following Table 20.
According to those described for the stress model, only zinc hyaluronate (M) was tested in the PAF-induced model. The zinc hyaluronate (M) strongly reduced the ulcerogenic effect of PAF, whereas sodium hyaluronate did not show such effect. Zinc chloride exhibited some protective action against PAF; this was, however, much less than that of the zinc hyaluronate associate (Table 20).
5. Inhibition of Acetic Acid Induced Chronic Qastric Ulcer
Female, RG Wistar rats weighing between 120-150 g were used. Before the surgery animals were deprived of food for 24 hours but received water ad libitum.
The chronic gastric ulcer was induced by the injection of 25 xcexcl/animal of 20% (v/v) acetic acid into the gastric wall under light ether anaesthesia according to the method similar to that described by Takagi et al. (1969). The oral treatment with the test compounds was started five days after the surgery and continued in a once-a-day regimen up to the 14th day. The animals were killed on the 15th day and their stomachs were dissected. The severity of ulceration was evaluated by the measurement of the diameter of the ulcers and the area affected was calculated.
The healing effect was expressed as differences in percentage between the control and test group values.
Acetic acid irritates the stomach wall and causes a well defined ulcer, which is very similar to the human ulcer both in its form as well as in its microscopical picture. The results are illustrated in the following Tables.